The Thermo Scientific 1-Step Human High-Yield In Vitro Translation (IVT) Kits enable the expression of functional proteins with 10 to 100 times greater yield per mL than other mammalian IVT systems.

The 1-Step High-Yield IVT System uses modified HeLa cell extracts to take advantage of the robust human translation machinery and generate functional full-length proteins. In this system, protein expression is performed in a proprietary dialysis device that allows a continuous supply of nucleotides, amino acids and energy generating substrates into the reaction while removing inhibitors of proteins synthesis. This continuous-exchange cell-free (CECF) system enables protein expression in an overnight incubation of up to 750 µg/mL. The complete mini-scale kit includes all the components required for transcription and translation of a recombinant gene, including an optimized expression vector.

Features of the 1-Step Human High-Yield Mini IVT Kit:

• High expression—up to 750 µg/mL of expressed protein• Reproducibility—low variability between experiments• Fast—express high levels of protein with 6 hours to overnight incubation• Functional—obtain functionally active proteins, including those containing disulfide bonds

1-Step Protein Expression:• Easy-to-use—transcription and translation is performed in one reaction step• Adapt for labeling—amenable to incorporation of heavy or unnatural amino acids

The 1-Step Human High-Yield IVT Kits are cell-free protein expression systems that provide all of the essential components required for transcription and translation. The kits are optimized with Accessory Proteins and Reaction Mixes that support protein synthesis using a DNA template. The advantages of using the 1-Step Human High-Yield IVT Kits over traditional in vivo expression systems include the ability to express toxic or insoluble proteins, easily perform protein labeling with modified amino-acids and reduce the time and cost of expressing human proteins in tissue culture cells. 1-Step Human IVT Kits are optimized for use with the pT7CFE1 expression vector that utilizes the T7 viral promoter and an EMCV Internal Ribosome Entry Site (IRES) to facilitate high levels of in vitro protein expression. Using a pT7CFE vector is critical for obtaining high expression levels. For added convenience, at family of pT7CFE vectors are available with tandem affinity tags for facilitate protein detection and purification. (For smaller scale and screening options, consider the 1-Step Human Coupled IVT Kit.)

The Thermo Scientific 1-Step Human High-Yield In Vitro Translation (IVT) Kits enable the expression of functional proteins with 10 to 100 times greater yield per mL than other mammalian IVT systems.

The 1-Step High-Yield IVT System uses modified HeLa cell extracts to take advantage of the robust human translation machinery and generate functional full-length proteins. In this system, protein expression is performed in a proprietary dialysis device that allows a continuous supply of nucleotides, amino acids and energy generating substrates into the reaction while removing inhibitors of proteins synthesis. This continuous-exchange cell-free (CECF) system enables protein expression in an overnight incubation of up to 750 µg/mL. The complete maxi-scale kit includes all the components required for transcription and translation of a recombinant gene, including an optimized expression vector.

Features of the 1-Step Human High-Yield Maxi IVT Kit:

• High expression—up to 750 µg/mL of expressed protein• Reproducibility—low variability between experiments• Fast—express high levels of protein with 6 hours to overnight incubation• Functional—obtain functionally active proteins, including those containing disulfide bonds

1-Step Protein Expression:• Easy-to-use—transcription and translation is performed in one reaction step• Adapt for labeling—amenable to incorporation of heavy or unnatural amino acids

The 1-Step Human High-Yield IVT Kits are cell-free protein expression systems that provide all of the essential components required for transcription and translation. The kits are optimized with Accessory Proteins and Reaction Mixes that support protein synthesis using a DNA template. The advantages of using the 1-Step Human High-Yield IVT Kits over traditional in vivo expression systems include the ability to express toxic or insoluble proteins, easily perform protein labeling with modified amino-acids and reduce the time and cost of expressing human proteins in tissue culture cells. 1-Step Human IVT Kits are optimized for use with the pT7CFE1 expression vector that utilizes the T7 viral promoter and an EMCV Internal Ribosome Entry Site (IRES) to facilitate high levels of in vitro protein expression. Using a pT7CFE vector is critical for obtaining high expression levels. For added convenience, at family of pT7CFE vectors are available with tandem affinity tags for facilitate protein detection and purification. (For smaller scale and screening options, consider the 1-Step Human Coupled IVT Kit.)

Thermo Scientific 1-Step Human In Vitro Protein Expression Kits enable the translation and post-transcriptional modification of full-length proteins from mRNA or plasmid templates with yields of up to 100 µg/mL per reaction.

The Human IVT Kits are unique HeLa cell lysate-based protein expression systems for in vitro translation or coupled transcription/translation reactions. Protein expression is performed in a single 90-minute reaction that can be extended for up to 6 hours with continued protein production up to 100 µg/mL when combined with the optimized pT7CFE1 Expression Vector. The Human IVT Kits can express functional enzymes, phosphoproteins, glycoproteins and membrane proteins for immediate use in studying protein interactions, performing rapid mutational analysis and measuring activity.

Features of the 1-Step Human Coupled IVT Kit:

• Functional—uses the human translational machinery to express active proteins• Convenient—perform transcription and translation in a single step• High performance—greater yields compared to rabbit reticulocyte in vitro translation• Reliable—express proteins that fail in rabbit reticulocyte systems

The 1-Step Coupled Human IVT Kit for DNA is a cell-free system using the cellular transcription and translation machinery from a modified HeLa cell line. The procedure is quick and easy and is an effective alternative to other protein expression methods. Simply add an appropriate expression construct to a mixture of HeLa cell lysate, Accessory Proteins, Reaction Mix and incubate at 30°C for 90 minutes to overnight.

The 1-Step Human IVT Kit for mRNA also uses the human protein translation machinery to produce functional protein. This kit is recommended for translation of mRNA transcripts containing an EMCV IRES element and mRNA stabilizing features designed into the pT7CFE1 expression vector.

The human in vitro translation system is robust and will express proteins from a variety of species including mammals, bacteria and protozoa. Benchmarking shows that the expression levels of functional proteins such as luciferase are much higher in the human system compared to either rabbit reticulocyte-based or E. coli-based systems. Furthermore, proteins expressed with the human in vitro translation system are not contaminated with substances that can interfere with downstream applications.

• Efficiently translates a wide variety of high molecular weight proteins and both capped and uncapped mRNAs• Accommodates large volume of template mRNA

How it worksThe lysate has been optimized for translation by the addition of an ATP-regenerating system, hemin, and calf liver tRNA. Included in the kit is an optimized Translation Mix (-Met or -Leu) for the translation of both capped and uncapped messages along with High and Low Salt buffers. These buffers can be used to maximize protein yield and activity from difficult-to-translate sequences. A report in Nucleic Acids Research (1) found that some commercial reticulocyte lysates had poor fidelity for initiation at the appropriate AUG initiator codon. The Retic Lysate IVT™ Kit was tested using Kozak's constructs and it demonstrated high fidelity for initiating at the proper AUG.

The Expressway™ Lumio™ Expression and Detection System takes advantage of the Lumio™ recognition sequence, a small, six amino acid sequence (Cys-Cys-Pro-Gly-Cys-Cys). The Lumio™ detection reagent binds the recognition sequence with high specificity and affinity, resulting in a bright fluorescent signal for real-time protein production analysis and immediate in-gel protein detection. In addition, Expressway’s specialized E. coli lysate, derived from a slyD mutant, eliminates nonspecific binding of the Lumio™ Green Detection Reagent to the endogenous SlyD protein and provides optimal background for detection of recombinant proteins (Figure 1). The Lumio™ Green Detection Kit is included in the system for real-time detection of protein synthesis as well as easy in-gel protein detection. The Lumio™ vectors (Figures 2 and 3) also feature:

• Efficiently translates a wide variety of high molecular weight proteins and both capped and uncapped mRNAs• Accommodates large volume of template mRNA

How it worksThe lysate has been optimized for translation by the addition of an ATP-regenerating system, hemin, and calf liver tRNA. Included in the kit is an optimized Translation Mix (-Met or -Leu) for the translation of both capped and uncapped messages along with High and Low Salt buffers. These buffers can be used to maximize protein yield and activity from difficult-to-translate sequences. A report in Nucleic Acids Research (1) found that some commercial reticulocyte lysates had poor fidelity for initiation at the appropriate AUG initiator codon. The Retic Lysate IVT™ Kit was tested using Kozak's constructs and it demonstrated high fidelity for initiating at the proper AUG.

The Expressway™ Mini Cell-Free Expression System utilizes an efficient coupled transcription and translation reaction to produce active recombinant protein. This system eliminates the time-consuming steps of cell-based protein production including transformation, cell culture, and expression optimization. In just two hours, you can produce protein suitable for an array of downstream applications and functional studies. The Expressway™ Mini Cell-Free Expression System offers flexibility in experimental design, allowing you to• Synthesize proteins from both circular plasmid and linear templates (e.g. PCR products)• Perform high-throughput synthesis of proteins • Express genes toxic to in vivo systems

How it works:The Expressway™ Mini Cell-Free Expression System provides all the components you need for optimal cell-free protein production. The kit includes an E. coli extract containing the cellular machinery required to drive transcription and translation. An IVPS (in vitro protein synthesis) reaction buffer and an amino acid module are also included in the kit to provide the required amino acids and an ATP-regenerating system. Simply mix the reaction buffer, amino acid mix, methionine (labeled or unlabeled), T7 Enzyme Mix, and your DNA template (containing a T7 promoter) with the E. coli extract. As the DNA template is transcribed, the 5’ end of the mRNA is bound by ribosomes and undergoes translation as the 3’ end of the template is still being transcribed (Figure 1).

Functional protein made easy:

The Expressway™ Mini Cell-Free Expression System has been optimized to produce high yields of full-length, functional protein from E. coli cell lysates (Figure 2). A proprietary feed buffer formulation is provided with the system for increased protein yields.

The Expressway™ Maxi Cell-Free E. coli Expression System uses an efficient, coupled transcription/translation reaction to produce milligram quantities of soluble, functionally active protein in 4-6 hours. The procedure can be performed in a single reaction tube and is easily scalable without the need for specialized equipment. The TOPO TA Cloning® expression vectors (5-min cloning with 95% efficiency) are provided for optimal expression results. In addition to all the advantages of an open expression system, Expressway™ Maxi technology provides a means to produce high levels of recombinant protein that may be easily detected and purified for various downstream applications.

Milligram level protein production within 4-6 hoursThe key to the Expressway™ Maxi technology is the unique formulation of the Feed Buffer that boosts the reaction productivity so that more than one milligram of protein is produced in a 2-ml reaction within 4-6 hours (Figure 1). The formulation of the lysate enables protein synthesis using either circular or linear templates.

High-throughput (HTP) compatible This system is also designed for HTP expression. One kit is good for 200 x 50-reactions, which can be accommodated by 2 x 96-well plates. Without feed buffer, such reactions need only two hour’s time. Once a positive expression is detected, scale-up protein production can be performed with the large Expressway™ cell-free format or in E. coli cell-based systems.

Optimized vectorspEXP5 TOPO® TA vectors are optimized for use in the Expressway™ Milligram system (Figure 2). They are designed to minimize additional amino acid sequences that may interfere with native protein folding and functionality. Features of these vectors include:

The Expressway™ Maxi Cell-Free E. coli Expression System uses an efficient, coupled transcription/translation reaction to produce milligram quantities of soluble, functionally active protein in 4-6 hours. The procedure can be performed in a single reaction tube and is easily scalable without the need for specialized equipment. The TOPO TA Cloning® expression vectors (5-min cloning with 95% efficiency) are provided for optimal expression results. In addition to all the advantages of an open expression system, Expressway™ Maxi technology provides a means to produce high levels of recombinant protein that may be easily detected and purified for various downstream applications.

Milligram level protein production within 4-6 hoursThe key to the Expressway™ Maxi technology is the unique formulation of the Feed Buffer that boosts the reaction productivity so that more than one milligram of protein is produced in a 2-ml reaction within 4-6 hours (Figure 1). The formulation of the lysate enables protein synthesis using either circular or linear templates.

High-throughput (HTP) compatible This system is also designed for HTP expression. One kit is good for 200 x 50-reactions, which can be accommodated by 2 x 96-well plates. Without feed buffer, such reactions need only two hour’s time. Once a positive expression is detected, scale-up protein production can be performed with the large Expressway™ cell-free format or in E. coli cell-based systems.

Optimized vectorspEXP5 TOPO® TA vectors are optimized for use in the Expressway™ Milligram system (Figure 2). They are designed to minimize additional amino acid sequences that may interfere with native protein folding and functionality. Features of these vectors include: